Anti-neoplastic, anti-viral and ribonucleotide reductase activity affecting pharmaceutical compositions and methods of treatment

ABSTRACT

A method for treating a human or non-human animal afflicted with malignant cells sensitive to parabactin or a pharmaceutically acceptable salt or complex thereof comprising administering to the animal an amount of parabactin effective to inhibit the proliferation of the malignant cells.

The invention described herein resulted in part from research conductedunder NIH Grant No. R01 AM29936. The United States Government hascertain rights in and to the claimed invention.

RELATED APPLICATIONS

This is a division of application Ser. No. 08/299,126,filed Sep. 2,1994, which is a division of application Ser. No. 08/124,557, filed Sep.22, 1993 (now U.S. Pat. No. 5,391,563, issued Feb. 21, 1995) which is adivision of application Ser. No. 07/993,620, filed Dec. 21, 1992 (nowU.S. Pat. No. 5,292,775, issued Mar. 8, 1994), which is a division ofapplication Ser. No. 07/645,644, filed Jan. 25, 1991 (now U.S. Pat. No.5,173,505, issued Dec. 22, 1992), which is a division of applicationSer. No. 07/313,734, filed Feb. 22, 1989 (now U.S. Pat. No. 5,128,353,issued Jul. 7, 1992), which is a continuation-in-part of applicationSer. No. 06/746,672, filed Jun. 20, 1985 (now abandoned).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to certain novel anti-neoplastic,anti-vital and ribonucleotide reductase catalytic activity affectingpharmaceutical compositions and methods of treatment.

2. Prior Art

Due to the critical role played by iron in the function of a variety ofbiological redox systems, e.g., ribonucleotide reductase, electrontransport proteins, iron flavorproteins, hydroperoxidase and oxygenases,life, with the possible exception of the lactobacilli, without iron isessentially unknown.

Microorganisms are particularly sensitive in their iron requirement andmany have evolved remarkable mechanisms for iron acquisition. Theysynthesize low molecular weight, virtually ferric ion specificchelators, i.e., siderophores which assist transport of exogenous ironinto the cell where it is utilized in supporting growth [Neilands, Ann.Rev. Biochem. 50, pp. 715-731 (1981); Bergeron, Chem. Rev. (in press)(1984); Emery, Metal Ions in Biological Systems, pp. 77-121 (1973)].Many of these chelators are relatively simple molecules consisting ofcatechol moieties (e.g., 2,3-dihydroxybenzoyl groups) attached tovarious amine backbones. All of these ligands bind iron tenaciously;e.g., parabactin, a siderophore synthesized by the plant pathogen,Paracoccus denitrificans [Tait, Biochem. 146, pp. 191-197 (1975)], formsa 1:1 metal complex with an iron formation constant in the order of 10⁴⁸moles/1.

Although certain microbes can interchangeably utilize the siderophoresof other prokaryotes, this ability is not universal. It has recentlybeen shown, for example, that parabactin cannot be utilized by largenumbers of bacterial pathogens [Bergeron et al, Antimicrob. Agents andChemo. 24, pp. 725-730 (1983)]. In fact, the siderophore exhibits potentbacteriostatic and fungistatic effects and appears to act by competingwith microbes for available iron.

Weinberg [Nutrition and Cancer 4, pp. 223-233 (1983) and PhysiologicalReviews 64, pp. 65-96 (1984)] has provided a compelling rationale foriron-withholding as a strategy against infection and neoplasia. Porteret al [Cancer Research 42, pp. 4072-78 (1982)] has shown that N¹,N⁸-dihydroxy-benzoylspermidine demonstrates significant cytotoxicity toL1210 cells (50% growth-inhibitory dose, 10 μM) and postulates that theactivity is probably attributable to its iron-chelating properties.However, not all iron chelators exhibit cytotoxicity.

It is an object of the present invention to provide certainanti-neoplastic, anti-viral, anti-psoriasis, anti-malarial andribonucleotide reductase activity affecting pharmaceutical compositionsand methods of treatment wherein the active agent is one of a smallclass of specific iron chelators.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions in unitdosage form adapted for administration to a human or non-human animalcomprising a) an anti-neoplastic, anti-viral, anti-psoriasis,anti-malarial or ribonucleotide reductase activity affecting, effectiveamount of a compound of the formula: ##STR1## Wherein: Z is ##STR2## Ris H or OH, x is 3 or 4,

y is 3 or 4, and

a is 1, 2or3,

or a salt or complex thereof with a pharmaceutically acceptable ion orligand, and b) a pharmaceutically acceptable carrier therefor.

The present invention also provides methods for treating human ornon-human animals afflicted with malignant cells comprisingadministering thereto an anti-neoplastic effective amount of a compoundof formula I or II.

The present invention also provides methods for treating human ornon-human animals afflicted with a ribonucleotide reductase dependentvital infection comprising administering thereto an anti-vital effectiveamount of a compound of formula I or II.

The present invention further provides a method for affecting thecatalytic activity of a ribonucleotide reductase in a human or non-humananimal, comprising administering thereto a ribonucleotide reductaseactivity affecting amount of a compound of formula I or II.

The present invention also provides a method for treating human ornon-human animals afflicted with psoriasis comprising administeringthereto an effective anti-psoriasis amount of a compound of formula I orII.

The present invention additionally provides a method for treating humanor non-human animals afflicted with malaria comprising administeringthereto an effective amount of a compound of formula I or II.

DETAILED DESCRIPTION OF THE INVENTION

Preferred among the compounds of formula I as active agents in thepharmaceutical compositions and methods of treatment of the presentinvention are those having the formula: ##STR3## wherein x and y havethe meanings set forth above.

Particularly preferred is the compound of formula III wherein x is 3 andy is 4.

Also preferred among the compounds of formula I are those having theformula: ##STR4## wherein x and y have the meanings set forth above.

Particularly preferred among the compounds of formula IV is that whereinx is 3 and y is 4.

Preferred among the compounds of formula II are those having theformula: ##STR5## wherein x and y have the meanings set forth above.

Particularly preferred is the compound of formula V wherein x is 3 and yis 4. Also particularly preferred are the compounds of formula V whereinx is 4 and y is 3 and where both x and y are 3.

The compounds of formulas I and II may be prepared according to themethods described in Bergeron et al, Synthesis, pp. 698-692 (1982);Bergeron et al, J. Org. Chem., 45, pp. 1589-1592 (1980); Bergeron et al,J. Med. Chem. 23, pp. 1130-1134 (1980); Bergeron et al, J. Org. Chem.46, pp. 4524-4529 (1983); Bergeron et al, J. Org. Chem. 48, pp.3432-3438 (1983); Bergeron et al, J. Org. Chem. 46, pp. 3712-3718(1981), the disclosures of each of which are incorporated by referenceherein.

The pharmaceutical compositions of the invention preferably contain apharmaceutically acceptable carrier suitable for rendering the compoundor mixture administrable orally as a tablet, capsule or pill, orparenterally or transdermally. The active ingredients may be admixed orcompounded with any conventional, pharmaceutically acceptable carrier.It will be understood by those skilled in the art that any mode ofadministration, vehicle or carrier conventionally employed and which isinert with respect to the active agent may be utilized for preparing andadministering the pharmaceutical compositions of the present invention.Illustrative of such methods, vehicles and carriers are those described,for example in Remington's Pharmaceutical Sciences, 4th Ed. (1970), thedisclosure of which is incorporated herein by reference. Those skilledin the art, having been exposed to the principles of the invention, willexperience no difficulty in determining suitable and appropriatevehicles, excipients and carriers or in compounding the activeingredients therewith to form the pharmaceutical compositions of theinvention.

The therapeutically effective amount of active agent to be included inthe pharmaceutical composition of the invention depends, in each case,upon several factors, e.g., the type, size and condition of the animal,the disorder to be treated, the intended mode of administration, thecapacity of the animal to incorporate the intended dosage form, etc.Generally, an amount of active agent is included in each dosage form toprovide from about 50 to about 500 mg, preferably from about 50 to about250 mg.

The active agent employed in the pharmaceutical compositions and methodsof treatment of the invention may comprise a pharmaceutically acceptablesalt or complex of the compounds of formulas I or II, e.g., sodium,potassium or other non-toxic metal salts, amine salts, etc.

The compound, compositions and method of the invention are useful forthe treatment of a wide variety of disorders. Exemplary of suchdisorders are leukemias, solid tumors and other cancers, ribonucleotidereductase dependent viruses, i.e., DNA viruses, e.g., herpes, etc.

Those skilled in the art will be aware that the amounts of the variouscomponents of the compositions of the invention to be administered inaccordance with the method of the invention to a patient will dependupon those factors noted above.

Generally, however, amounts of active agent are administered to providedosages thereof from about 50 to about 500 mg/kg, preferably from about50 to about 250 mg/kg, the frequency of administration and duration oftreatment being dependent upon the type and nature of the animal anddisorder treated.

The invention is illustrated by the following non-limiting example.

EXAMPLE

Murine L1210 leukemia cells were maintained in logarithmic growth as asuspension culture in RPMI 1640 containing 2% HEPES-MOPS buffer and 10%fetal calf serum as described by Porter et al, Science 219, pp.1083-1085 (1983). Cultures were treated while in logarithmic growth (0.5to 1×10⁵ cells/ml) with the test compounds at concentrations rangingfrom 10⁻⁶ to 10² M. After 24 to 48 hours, cells were counted byelectronic particle analysis and viability determinations with trypanblue. Total iron content of the medium was determined by atomicabsorption analysis.

All compounds tested were initially screened for anti-viral activityagainst herpes simplex I, KOS strain or vesicular stomatitis virus inCV-1 monkey kidney cells using a methyl cellulose disk overlay assay asdescribed by Schroeder et al, J. Med. Chem. 24, pp. 1078-1983 (1981).The inhibition of virus replication was determined quantitatively bymeasuring the amount of virus produced by infected cells during a singlecell cycle of infection, in the presence of the compound tested. CV-1cells were infected with 20 PFU/cell of herpes simplex virus. One hourpost infection, cells were rinsed with Modified Eagle's Medium andexposed for 30 min. to a medium composed of 1% anti-herpes rabbitantiserum to neutralize unpenetrated virus. The cells were rinsed oncewith medium and then cultured with the test compound. A baseline usedfor measuring net virus production was obtained by harvesting andfreezing one sample which was untreated 4 hours after infection. Theremaining samples were collected and frozen 18-20 hours post infection.Virus was titrated in CV-1 cells by a plaque assay as described byHughes et al, J. Virol. 16, pp. 275-283 (1975). In studies designed toprevent the anti-viral activity of the chelators, FeCl₃ at variousconcentrations was added to medium containing a fixed concentration ofthe test compounds and the anti-vital assay performed as describedabove.

The partition equilibrium values for octanol and phosphate bufferedsaline were determined for certain of the test compounds. The compoundswere added in nitrogen saturated methanol at the same concentration tothree acid washed test tubes and the alcohol evaporated leaving a thinfilm of compounds coating the interior of the tube. Next, 5 ml each ofdegassed phosphate buffered saline (pH 7.4) and n-octanol were added.The tubes were sealed with Teflon caps under argon and rotated at 6 rpmat 27° C. for 12 hours. Samples were removed from the n-octanol and PBSlayers and measured for the various compounds by observing the samplesabsorbance at the appropriate wavelength; parabactin, 333 nm; GABA, 330nm; Compound II, 329 nm; and dihydroxybenzoic acid, 319 nm. Absorbancevalues were compared to Beer's Lambert's plots generated for eachcompound.

The effectiveness of the test compounds as inhibitors of cell growth orvital replication was assessed according to the drug concentrationrequired to reduce cell growth or vital plaque formation by 50% (IC₅₀).Against L1210 leukemia, all of the spermidine catecholamides were activein the micromolar range with parabactin and GABA being the mosteffective at 2 μM (see Table I). The catecholamides were even moreactive against the DNA virus, herpes simplex type I, inhibitingreplication at concentrations ranging from 0.4 μM (parabactin) to 55 μM.By contrast, they were totally inactive against the RNA virus, vesicularstomatitis, at concentrations up to 1 μM.

                  TABLE I                                                         ______________________________________                                        In Vitro Antileukemic and Antiviral Activity                                  of Spermidine Catecholamides and Related Compounds                                             Anti-                                                                  Forma- leukemic Antiviral Activity                                              tion     Activity Herpes Vesicular                                Polymine    Constant (IC.sub.50,                                                                            simplex I                                                                            stomatitis.sup.+                         Derivative  (moles/l)                                                                              48 hr)   (IC.sub.50)                                     ______________________________________                                        N-benzylspermidine                                                                        0         4.0 mM    1 mM 1 mM                                     2,3-dihydroxy-                                                                            10.sup.36                                                                               2.8 mM    1 mM 1 mM                                     benzoic acid                                                                  (DHBA)                                                                        N.sup.1,N.sup.8 -bis(2,3-                                                                 10.sup.40                                                                              14.0 μM                                                                             55.0 μM                                                                           1 mM                                     dihydroxybenzoyl)-                                                            N.sup.4 -threonyl-                                                            spermidine                                                                    N.sup.1,N.sup.8 -bis(2,3-                                                                 10.sup.40                                                                               7.0 μM                                                                             32.0 μM                                                                           1 mM                                     dihydroxybenzoyl)-                                                            spermidine                                                                    (Compound II)                                                                 N.sup.1,N.sup.8 -bis(2,3-                                                                 10.sup.45                                                                               2.0 μM                                                                             18.0 μM                                                                           1 mM                                     dihydroxybenzoyl)-                                                            N.sup.4 -(4-[2,3-di-                                                          hydroxybenzamido]-                                                            butyryl)spermidine                                                            (GABA)                                                                        N-[3-(2,3-dihydroxy-                                                                      10.sup.48                                                                               2.0 μM                                                                               0.4 μM                                                                          ND**                                     benzamido)propyl]-                                                            N-[4-(2,3-dihydroxy-                                                          benzamino)-                                                                   butyryl]-2-(2-hy-                                                             droxyphenyl)trans-                                                            5-methyl-oxazoline-                                                           4-carboxamide                                                                 (Parabactin)                                                                  N-[3-(2,3-dihydroxy-                                                                      10.sup.48                                                                               2.0 μM                                                                             ND**   ND**                                     benzamido)propyl]-                                                            1,3-bis(2,3-di-                                                               hydroxyphenyl)-                                                               trans-5-methyl-2-                                                             oxazoline-4-carbon-                                                           amido]propane                                                                 (Vibrobactin)                                                                 ______________________________________                                         **Not determined                                                              .sup.+ Estimated by methyl cellulose disk overlay assay [Schroeder et al,     J. Med. Chem. 24, pp. 1078-1083 (1981).                                  

The compound appears to be non-toxic to the monkey kidney cellmonolayers used in the plaque inhibition assay at concentrations whichfully inhibited herpes replication. In fact, when resting cellmonolayers were treated for 24 hours with 100 μM Compound II and thenreplaced in drug-free media, their growth was identical to that ofuntreated cells.

When the compounds were ranked according to their estimated iron bindingconstants (Table I), the order generally paralleled both theanti-leukemic and anti-herpetic activities of the compounds. The simplebidentate parent ligand DHBA displayed minimal activity, while N⁴-benzylspermidine, which has no iron chelating potential, was virtuallyinactive in either system. This suggests that the spermidine moiety ofthe siderophores was not responsible for their activities. Furthermore,the effects of the ligands could be prevented by addition of ferricchloride, supporting the idea that iron chelation is the source ofactivity. Both the anti-leukemic activity and the anti-herpetic activityof Compound II were fully attenuated by the inclusion of exogenous ironin the incubation media (Table II). Similar results were also obtainedwith parabactin in the herpes replication assay.

While not wishing to be bound by any theory as to the .mechanism of theaction of the active ingredients of the invention, it is hypothesizedthat the activities of the compounds are not only related to the abilityto sequester iron but to the ability of nonchelated siderophores todiffuse across the plasma membrane. As has been shown by Raymond andco-workers, [J. Amer. Chem. Soc., 100, pp. 5362-5370 (1978)], the rateof removal of iron from transferrin by catecholamide iron ligands isquite slow. Considering that a vast majority of iron in tissue culturemedia (8 μM) is bound to transferrin, it is reasonable to postulate thatthe catecholamides can exist for some time in the media in a free state,during which time they may diffuse into cells at rates associated withfavorable partition coefficients.

                  TABLE II                                                        ______________________________________                                        Prevention of the Anti-leukemic and Anti-                                     herpetic Activities of Compound II with Fe (III)                                         Compound II   (FcCl)                                               System     (μM)       (μM) % Control                                    ______________________________________                                        L1210 growth                                                                             10            0       23                                                      10            4       64                                                      10            8       96                                                      10            12      104                                          Herpes replication                                                                       100           0       0                                                       100           9       20                                                      100           19      53                                                      100           38      138                                          ______________________________________                                    

The partitioning of various chelators between n-octanol and phosphatebuffered saline is indicated in Table III. It should be noted that theratios of ligand partition coefficients (catecholamide/DHBA) are closerto the ratios of the chelator's anti-leukemic activity than are theratios of the binding constants (Table I). The best example is seen whencomparing the activity of parabactin with DHBA. Although parabactin is10¹² times more effective at binding iron than DHBA, it is only 10³times more active as an antineoplastic. However, the ratio of theirpartition constants between octanol and phosphate buffered saline ismuch closer to their biological activity ratios 10¹.7. Similar resultsare obtained when comparing the activities of each of the otherchelators relative to their partition coefficients indicating that cellpenetration as well as ion-chelation potential may determinecatecholamide activities.

                  TABLE III                                                       ______________________________________                                        Partition Coefficients and G°.sub.transfer Determined                  in: Phosphate Buffered Saline (PBS) pH = 7.40;                                n-octanol Mixture                                                                                    Conc.n-Octanol                                         Chelator  Solubility in PBS                                                                          Conc. PBS   G°.sub.t .sup.K cal.sup.M-I         ______________________________________                                        DHBA      1.30 × 10M                                                                           0.07        +1.57                                      Compound II                                                                             2.55 × 10M                                                                           0.97        -0.04                                      Parabactin                                                                              1.29 × 10M                                                                           32.44       -2.06                                      ______________________________________                                    

The selective effect on the iron chelators on DNA (but not RNA) virusreplication strongly suggests that they are affecting ribonucleotidereductase, a rate-limiting enzyme in DNA synthesis which catalyzes theconversion of ribonucleotides to deoxyribonucleotides. The enzymeconsists of two subunits, one of which is heme-containing and essentialto function [Thelander et al, Ann. Rev. Biochem. 48, pp. 133-158(1979)]. Precedent for such drug action is provided by the anticanceragent, hydroxyurea, which inhibits cell growth by interfering withribonucleotide reductase via a free radical scavenger mechanisminvolving the iron moiety of the enzyme [Thelander et al, Supra; Lanikenet al, J. of Virol. 41, pp. 893-900 (1979)].

It is well-known that malignant cells require DNA to divide and that ifsuch a cell cannot generate sufficient DNA, it will eventually die. Theenzyme ribonucleotide reductase is required to manufacture thedeoxyribonucleotide precursors in the malignant cell for DNAmanufacture. The compounds of formulas I and II above inhibitribonucleotide reductase and therefore are cytotoxic for a wide varietyof malignant cells. [Ganeshagurn et al, Biochemical Pharmacology, Vol.29, pp. 1275-1279 (1980); Bergeron et al, Biochem. and Biophys. Res.Comm., Vol. 121, pp. 848-854 (1984); Bergeron et al, J. Med. Chem., Vol.23, pp. 1130-1133 (1980) ; Sato et al, Cancer Research, Vol. 41, pp.1637-1641 (1981); Elford Biochem. and Biophys. Res. Comm., Vol. 33, pp.129-135 (1968); Lederman et al, Blood, Vol. 64, pp. 748-758 (1984)].

Most importantly, the compounds of the invention show little, if any,toxicity to normal cells, further underscoring their value as broadspectrum anti-neoplastic agents.

It should also be noted that the active ingredients of the presentinvention are more effective anti-neoplasts and anti-vital agents thanCompound II which was previously disclosed by Porter et al, supra. Thedifference in activity is associated with the enhanced lipophilicity andiron binding ability of the active agents of the invention.

The IC₅₀ values at which the present chelators inhibited growth of L1210cells and replication of herpes virus are in the range ofanti-neoplastic and anti-vital agents being used clinically. Forexample, the anti-herpetic agents,2-fluoro-5-iodo-I-B-D-arabinofuranosylcytosine (known as FIAC) andacyclovir, have IC₅₀ values of 0.6 and 40 μM, respectively, in an invitro system using the same herpes strain but a slightly different(veto) monkey kidney cell line [DeClercq, Antimicrob. Agents and Chemo.212, pp. 661-663 (1982)].

The compounds of formula I and II have also been found to be effectiveanti-psoriasis agents. As described hereinbefore, these compoundsinhibit cellular proliferation at the ribonucleotide reductase level.Ribonucleotide reductase is an iron-dependent enzyme controlling therate limiting step of DNA synthesis, and is greater than 95% inhibitedby the above compounds at micromolar concentrations. Because psoriatictissue is rapidly proliferating tissue and is highly dependent onribonucleotide reductase, the compounds described herein are activeanti-psoriasis agents.

Research leading to the present invention was supported by Grants NIAMDD(AM-29936), CA-33321 and CA-22153 from the National Cancer Institute andby the Veterans Administration. The U.S. Government has certain rightsin this invention.

The compounds of formula I and II have also been found to be effectiveanti-malarial agents. While not wishing to be bound by any theory as tothe anti-malarial mechanism of the compounds, it is hypothesized thatthe activity is somehow predicated on the heavy metal chelatingproperties of the compounds. The following example illustrates theanti-malarial aspects of the invention.

EXAMPLE 1

The anti-malarial activity of the compounds listed in Table IV weredetermined using the method of Scheibel et al, Mol. Pharmacol., 20, pp.218-223 (1981).

In this study, Plasmodium falciparum were grown in petri dishes usingthe conventional candle jar technique (Jensen, J. B., and W. Trager.Plasmodium falciparum in culture: use of outdated erythrocytes anddescription of the candle jar method. J. Parasitol. Vol. 63, pp. 883-886(1977). Parasites were grown for 24 hours in 1.5 ml petri dishes in acandle jar before exposure to the chelators of interest. The Plasmodiumfalciparum were next exposed to various concentrations of the ligands,and ED₅₀ values determined after two and three days of exposure.Although all of the ligands were active, parabactin was the most activeat day two with an ED₅₀ of 2.6 μM while vibriobactin was the most activechelator at three days with an ED₅₀ of 1.8 μM.

                  TABLE IV                                                        ______________________________________                                        Concentration (in μM) required to reduce in vitro                          growth of P. falciparum 50% (ED.sub.50) after                                 exposure for 2 days and 3 days                                                               Day 2 Day 3                                                    ______________________________________                                        Vibriobactin     4.5     1.8                                                  Parabactin       2.6     2.3                                                  Compound II      4.5     3.7                                                  GABA             5.1     4.3                                                  ______________________________________                                    

As is apparent from the results in Table IV, all of the compounds areactive anti-malarial agents.

I claim:
 1. A method for treating a human or non-human animal afflictedwith malignant cells comprising administering thereto an antineoplasticeffective amount of a compound of the formula: ##STR6## Wherein: Z is##STR7## R is H or OH, x is 3 or 4,y is 3 or 4, and a is 1, 2 or 3, orapharmaceutically acceptable salt or complex thereof.
 2. The method ofclaim 1 wherein said compound has the formula: ##STR8## wherein x and yhave the meanings set forth in claim
 1. 3. The method of claim 2 whereinx is 3 and y is4.
 4. The method of claim 1 wherein said compound has theformula: ##STR9## wherein x and y have the meanings set forth inclaim
 1. 5. The method of claim 4 wherein x is 3 and y is4.
 6. Themethod of claim 1 wherein said compound has the formula: ##STR10##wherein x and y have the meanings set forth in claim
 1. 7. The method ofclaim 6 wherein x is 3 and y is4.
 8. The method of claim 6 wherein x is4 and y is3.
 9. The method of claim 1 comprising administering to saidanimal an anti-leukemic effective amount of said compound.
 10. Themethod of claim 1 wherein said compound is parabactin.